Glucosamine synthetase from Escherichia coli: purification, properties and glutamine-utilizing site location
L-Glutamine: D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH/sub 3/-dependent activity and did not catalyze the reverse reaction. The enzyme has a K/sub m/ of 2 mM for fructose 6-phosphate, a K/sub m/ of 0.4 mM for glutamine, and a turnover number of 1140 min/sup -1/. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH/sub 2/-terminal residue. The enzyme was inactivated by the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with (6-/sup 14/C)DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH/sub 2/-terminal cysteine residue as shown by Edman degradation.
- Research Organization:
- ENSCP, Paris, France
- OSTI ID:
- 5964553
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:7; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINES
AMINO ACIDS
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBOHYDRATES
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CROSS-LINKING
ENZYMES
ESCHERICHIA COLI
GLUCOSAMINE
HEXOSAMINES
HEXOSES
INACTIVATION
KINETICS
LABELLED COMPOUNDS
LEUCINE
LIGASES
MICROORGANISMS
MOLECULAR WEIGHT
MONOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
POLYMERIZATION
PURIFICATION
REACTION KINETICS
SACCHARIDES